The goal of this project is to investigate a potential mechanism by which amyloid- peptides (A) form aggregates in the brain. This is directly relevant to the pathogenesis of Alzheimer's disease (AD). In vitro studies have shown that A forms aggregates only at concentrations that are more than one thousand times higher than those measured in cerebrospinal fluid and in brain tissue. Even so, A plaque formation is characteristic for AD and genetic evidence demonstrates that mutations that increase A aggregation propensity lead to early onset AD. This apparent contradiction suggests that there must either be: 1) high local concentrations of A somewhere in the brain, or 2) cellular and/or biochemical mechanisms which lower the concentration required for A to aggregate. Growing evidence suggests that A can accumulate in intraneuronal vesicles well before the first appearance of amyloid plaques. Our preliminary data suggest that A can be endocytosed and trafficked through the endo/lysosomal pathway. Moreover, we have found that A is concentrated in late endo/lysosomes to levels that would support A aggregation. This occurs even when low, physiologically relevant concentrations of A are added to the outside of the cell. Also, we know that certain features of the endo/lysosomal environment, such as low pH, favor A aggregation. This makes the endo/lysosomal pathway particularly attractive for nascent A aggregate formation in vivo. The hypothesis to be tested in this work is that the earliest stages of A aggregation in vivo occur within neural cells during endo/lysosome maturation. In this proposal, we will investigate A uptake from the extracellular space and the effect of trafficking along the endo/lysosomal pathway on its aggregation state. We will use modern fluorescence spectroscopic and microscopic methods to develop a detailed time course of the uptake, trafficking, and aggregation of A along the endo/lysosomal pathway. These experiments will be combined with modern cell biology tools to interrogate how specific environmental factors of the endo/lysosomal pathway contribute to the aggregation of A. We speculate that endocytosis and trafficking of A along the endo/lysosomal pathway represents a normal pathway for quality control and maintenance of extracellular A levels. However, characteristics of the endo/lysosomal environment, such as low pH and locally high protein concentrations, favor A aggregation. It is quite possible that the balance between A aggregation and degradation along the endo/lysosomal pathway is a critical determinant in AD pathogenesis.